JP4511983B2 - Multiple sample automatic processing system and multiple sample automatic processing method - Google Patents

Description

  The present invention relates to a multiple sample automatic processing system and a multiple sample automatic processing method for processing a sample through a plurality of processing steps.

  In many fields, such as sample analysis, it is necessary to perform a plurality of processes on a sample in stages. For example, in proteome analysis, a protein sample (gel) is converted into a peptide mixture and supplied to a mass spectrometer. As pretreatment, protein gel band destaining, gel band drying, reductive alkylation reaction, enzyme digestion reaction, etc. Must be performed in order.

Toshiaki Isobe, Nobuhiro Takahashi / Edited “Proteome Analysis, Advanced Protein Expression and Functional Analysis, Genome and Drug Discovery Research” (Yodosha) Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar 1; 68 (5): 850-8.

  If it demonstrates regarding peptide mixture preparation, it can also prepare manually and can also prepare automatically using a commercially available automatic preparation apparatus. However, at present, each case has advantages and disadvantages. In the case of manual preparation, since processing is usually performed one sample at a time, proceed while confirming the reaction status of each individual sample, and unreacted samples must be reacted appropriately each time to prevent the occurrence of defective samples. However, due to human preparation, sample misunderstanding due to carelessness of the operator may occur, and depending on the level of skill, the reproducibility of the operation may be low and processing may be delayed or conditions may not be constant. , And contamination problems where proteins from the worker enter the sample. On the other hand, since the automatic preparation device is prepared without human intervention, it is excellent in terms of being capable of simultaneous processing in large numbers, preventing human error, and preventing contamination. Since there is no monitoring function, there is a problem that the defect rate eventually increases. Further, in an automatic preparation apparatus that performs a large number of simultaneous processes, even if a reaction monitoring mechanism is provided to monitor the reaction status, it is difficult to individually perform error processing.

  In view of such problems, the present invention completely automates the process, eliminates human error in many sample processing, reduces contamination, enables quantification of products, and suppresses the defect rate. An object of the present invention is to provide a system that can

  In the present invention, the object is achieved by fully automating the processing steps, providing a reaction monitoring function in each step, extracting an unreacted sample, and causing a re-reaction. In the present invention, for example, when it is possible to place 8 × 12 sample containers in the sample container storage area, a maximum of 96 sample containers can be continuously processed. At this time, eight sample containers are set as one unit, and each unit is transported and supplied to each dispensing / aspirating device and each reaction tank. In addition, after completion of each reaction / treatment step, the sample container is returned to the original position of the sample container plate. In order to ensure the quantitativeness and certainty of the reaction and processing of the sample, the reaction state is monitored by the reaction monitoring sensor as necessary, as well as the stability of the dispensing and suction of the processing solution (quantitative dispensing and complete suction). The sample determined to be defective by monitoring is returned to a predetermined process location and reacted and processed again.

  That is, the multiple sample automatic processing system of the present invention includes a sample container storage place where a plurality of sample containers are placed, a dummy container place where a dummy container is placed, and a first processing unit that performs different processing on the samples in the container. And a second processing unit, an inspection unit for inspecting a sample in the container, a container transport unit for transporting the container between each container storage place, the processing unit and the inspection unit, and a control unit, Each part is controlled, and the first processing unit performs processing with n (n is a positive integer of 2 or more) containers as one unit, and after that processing, there is a sample that fails the inspection by the inspection unit When a defective container is added to a container containing the rejected sample and a set of n containers is processed, the dummy container is added to the container containing the sample that has passed the inspection. The container made into a set is processed in the second processing unit.

  The container transport unit preferably includes a first container holding unit that holds n containers simultaneously and a second container holding unit that holds one container. The first processing unit and the second processing unit include a dispensing unit that injects or sucks the processing liquid into a set of n containers. The first processing unit and the second processing unit can be shared by the dispensing unit, and the dispensing unit may be provided in the container transport unit.

  In the multiple sample automatic processing method of the present invention for processing a plurality of samples through a plurality of processing steps, the first processing unit includes n (n is a positive integer of 2 or more) containers as a set. When there is a process that performs a process on the inner sample, a process that performs a first inspection on a sample that has been processed by the first processing unit, and a sample that fails the first inspection Adding a dummy container to the container containing the rejected sample to form a first container unit composed of n containers, and performing a defect treatment on the first container unit; A step of performing a second inspection on a sample after defect processing, and a step of forming a second container unit composed of n containers by adding a dummy container to a container containing a sample that has passed the second inspection. And add a dummy container to the container containing the sample that passed the first inspection and add n Forming a third container unit consisting of the container, in the second processing unit, and a step of performing processing on the sample and the third sample in the container unit of the second container unit.

  According to the present invention, it is possible to automate the processing of a sample that passes through a plurality of processing steps to process a large number of samples quickly and reproducibly, prevent contamination, and the sample defect rate due to incomplete processing. Can be reduced.

  Embodiments of the present invention will be described below. Here, the present invention will be described with reference to an embodiment of a process for preparing a digested peptide mixed sample for processing gel bands or spots of proteins separated by electrophoresis and analyzing them by liquid chromatography mass spectrometry. The sample processing system of this example is intended to automatically perform a chemical reaction and an enzymatic reaction on a protein sample for proteome analysis, convert it into a peptide mixture, and supply it to a mass spectrometer. The protein spot (gel piece) obtained by electrophoresis is placed in a sample container (capacity 700 μl), and subjected to reaction / treatment such as destaining, reductive alkylation, in-gel digestion and desalting / concentration, etc. to the mass spectrometer. To supply. In addition, this invention is not limited to the Example demonstrated here, It can apply to the processing method generally which processes a several step | paragraph with respect to a some sample.

  FIG. 1 is a schematic diagram showing the overall configuration of a sample processing system according to the present invention. This sample processing system includes a sample container storage 11 for holding a test tube transparent container containing a sample, a dummy container storage 12 for holding a test tubular transparent container (dummy container) containing no sample, and an inspection described later. In the bottom of the transparent container, there is a defective sample storage place 13, a test container storage place 51, a reagent bottle storage place 50 containing a solid reagent, and a transparent container. Bar code reader 15 for reading a two-dimensional bar code for identifying individual samples, a vibrating thermostat 16, a vacuum centrifuge 17, a thermostatic chamber 18a, an ice tub 18b, a light shielding bath 19, a CCD camera for inspection Has an inspection station 20 in which is placed. Further, a pipette tip storage place 60 for placing disposable pipette tips, and a pipette tip disposal place 61 for discarding used pipette tips are provided.

  Above these, a transport head 23 for transporting a plurality of containers, for example, eight containers as a unit to each part is installed. The transport head 23 can move in the X direction along the ball screw 22, and the ball screw 22 can move in the Y direction along the rails 21a and 21b. As a result, the transport head 23 can transport the container to any place in the XY directions. The dispensing station 14 has eight nozzles connected to the reagent container and the destaining liquid container, and injects the treatment solution into one unit of the container that has been transported by the transport head 23, that is, eight containers. Or the solution can be aspirated from the container. By switching the processing liquid container connected to the nozzle, the processing liquid necessary for processing is selected and supplied from the nozzle to each container. Each unit of the apparatus is controlled by the control unit 24.

  The processing unit for processing the sample in the container includes a plurality of facilities shown in FIG. 1 such as a dispensing device and a thermostatic chamber for injecting and sucking a processing liquid into the container, and a dispensing device and a vibrating thermostatic chamber. Composed. The dispensing device may be provided individually in each processing unit, or may take a form shared by a plurality of processing units. In this embodiment, a plurality of processing units share one dispensing device. Further, the dispensing device may be fixedly provided on the device or may be provided on the transport head.

  2A and 2B are explanatory views showing an example of the transport head. FIG. 2A is a schematic front view, and FIG. 2B is a schematic bottom view. Below the transport head 23, a rod 31 having a container gripping portion 32 at the tip protrudes. The lower end of the container gripping portion 32 swells in a direction perpendicular to the rod axis, and fits into a recessed portion of the container lid described later. A hollow outer cylinder 52 having a diameter larger than that of the container gripping portion 32 is provided outside the rod, and can move up and down independently of the rod. The eight rods 31 constitute one unit to form the first container transport unit 33, and the first container transport unit 33 moves up and down at the same time as indicated by arrows. Similarly, the transport head 23 includes a rod having a container gripping portion and an outer cylinder at the tip, but also includes a second container transport portion 34 that can transport only one container independently. The eight outer cylinders of the first container transport section 33 are also moved up and down at the same time, with eight serving as one unit.

  The dispensing device for dispensing the processing liquid into the container may be fixedly provided in the dispensing station 14 or provided in the transport head, and the transport head moves to the dispensing station during dispensing. May be dispensed. FIG. 3 is an explanatory view showing an example of a transport head provided with a dispensing device, in which FIG. 3A is a schematic front view, and FIG. 3B is a schematic bottom view. The transport / dispensing head 23 is composed of a transport head portion and a dispensing head portion, which may be moved up and down simultaneously or independently.

  A rod 31 having a container gripping portion 32 at the tip protrudes below the transport head portion. The distal end of the container gripping portion 32 swells in a direction perpendicular to the rod axis, and fits into a concave portion of the container lid described later. A hollow outer cylinder 52 having a diameter larger than that of the container gripping portion 32 is provided outside the rod, and can move up and down independently of the rod. The eight rods 31 constitute one unit to form the first container transport unit 33, and the first container transport unit 33 moves up and down at the same time as indicated by arrows. Similarly, the transport head portion is composed of a rod having a container gripping portion and an outer cylinder at the tip, but also has a second container transport portion 34 capable of independently transporting only one container.

  A dispensing head 55 to which a pipette tip 57 can be attached at the tip protrudes below the dispensing head. Eight dispensers constitute one unit to form a first dispenser 58, and the first dispenser 58 moves up and down simultaneously as indicated by an arrow. Similarly, the dispensing head portion is composed of a dispenser capable of mounting a pipette tip 57 at the tip, but also has a second dispensing portion 59 capable of independently dispensing and sucking only one container. . The dispenser is provided with a hollow tip detachment outer cylinder 56 that can move up and down independently with respect to the dispenser head 55.

  FIG. 4 is an explanatory diagram showing how the container 31 is transported and the container lid is opened and closed by the rod 31 having the container gripping portion 32 at the tip. FIG. 4A shows a state in which the transport head is positioned above a container 41 with a lid placed on a sample container storage place and the rod 31 having a container gripping portion 32 at the tip is lowered from the transport head. Yes. FIG. 4B shows a state in which the container gripping portion 32 provided at the tip of the rod 31 is inserted and fixed in the concave portion of the lid 53 fitted to the opening of the container 41.

  FIG. 4C shows a state where the lid 53 of the container is opened. The holding pin 54 is normally opened by a spring or a magnetic force as shown in FIG. 4A. However, the container 41 can be fixed by moving inward by air pressure or the like and pressing the lower part of the container. In this state, the lid 53 can be removed from the container 41 by raising the transport head while fixing the lid 53 to the rod 31 as shown in FIG. FIG. 4 (d) shows a state in which the rod 31 is raised in a state where the container gripping portion 32 is fixed to the concave portion of the lid 53 fitted into the opening of the container 41. The container 41 is pulled up by the rod 31 with the container gripping part 32 fitted in the recess of the lid 53. By moving the transport head in this state, the container is transported.

  FIG. 4E shows a state in which the container that has been transported is placed. After transporting to the desired position, ground. Thereafter, the rod outer cylinder 52 is lowered, and the rod 53 is lifted while the lid 53 is pressed, whereby the lid 53 can be removed from the transport head 23. At this time, the lid 53 remains fitted in the opening of the container 41.

  The structure of the transport head shown in FIGS. 2 and 3 or the container transport method shown in FIG. 4 is merely an example, and the specific structure and transport method for transporting the container are structures other than those shown here. Or a method may be used.

  In the case of the transport head equipped with the dispensing device shown in FIG. 3, when the treatment liquid has been dispensed or aspirated, the control unit 24 moves the transport head above the pipette tip disposal site 61, where it is used for chip detachment. The outer cylinder 56 is lowered, and the pipette tip attached to the dispenser head 55 is pushed down to be removed and discarded in the pipette tip disposal place 61. In mounting the pipette tip, the control unit 24 moves the transport head above the pipette tip storage 60 and positions the dispenser head 55 so as to align with the pipette tip placed in alignment with the pipette tip storage 60. To do. Then, the pipette tip is attached to the dispenser head 55 by lowering the dispenser head portion and inserting the dispenser head 55 into the opening at the upper end of the pipette tip. Thereafter, the dispensing head unit is raised, the transport head is moved to the dispensing station, and a dispensing operation is performed.

  FIG. 5 is a flowchart showing an outline of processing in each processing step. The sample that has been processed in the previous step is processed in units of a predetermined number of containers (S11). When the processing is completed, the containers of the respective samples are individualized (S12), and individually inspected whether necessary processing is completed (S13). Samples that pass the inspection are grouped into one unit (S15) and transferred to the next processing step. Samples that have been rejected by the inspection are unitized between the rejected samples (S17), and are sent to defect processing (S18). When the defect processing is completed, the process is personalized again (S12) and is inspected (S13). The number of defective processes is set in advance, and a container containing a sample that does not pass the inspection even if the defective process is performed a predetermined number of times is registered as a defective product, and the subsequent process is not performed. In the unitization of S15 and S17, dummy containers are added to match the number of containers to the number of one unit (eight in this embodiment). Further, the individualization of S12 is a process required for individually inspecting each sample, and since the individual sample can be inspected in S13 even when the container is unitized. If there is, the step of S12 may be omitted.

  FIG. 6 is a conceptual diagram visually showing the flowchart of FIG. A container containing a sample is processed in units of a predetermined number of containers. Thereafter, one unit of the container is individualized, and the processing state of each container is inspected. At this time, as shown in the figure, it is assumed that seven containers marked with ◯ passed the inspection, but one container marked with x failed. In this case, the container is divided into a passed container and a failed container, and one dummy container is added to the passed container to make eight, and a failed container is added with seven dummy containers to eight. To do. In this way, the unit is formed, and the rejected unit is sent to the defective processing. The container that finally passed the inspection due to the defective processing is unitized again, and is sent to the next processing step together with the previously passed unit.

  FIG. 7 is an explanatory diagram showing in more detail the flow of a container containing a sample that has passed the inspection and a container containing the unacceptable sample.

  Samples processed in units are inspected to determine whether individual samples in the unit are normal samples or defective samples. The defective sample is removed from the unit, and a dummy container is inserted in the place where the defective sample is present to form a normal sample unit. On the other hand, a defective sample taken out from the unit is similarly added with a dummy container to form a unit, and the unit is subjected to defect processing. When defective processing is completed, the inspection is performed again, and a sample that still fails the inspection is taken out of the unit and transferred to the defective sample storage. In addition, a dummy container is added at the place where the defective sample is taken out to form a normal sample unit. The two normal sample units thus obtained are processed in the next processing step. When the final processing step is completed, the dummy container is removed from the unit, and the sample that has been subjected to various types of processing normally is stored in the normal sample storage.

  6 and 7 show an example in which a dummy processing unit is added to a defective sample to form a defective processing unit for each unit that has been inspected. However, if time for reaction is allowed, if the total number of samples is 96, all 96 samples (or any intermediate number such as 48) will be inspected and then rejected from each unit. A processing method is also possible in which defective sample units of 8 units are formed by taking out and collecting them (if the number exceeds 8, the first 8 units are unitized and a dummy is added to the rest).

Next, a specific example of processing will be described. The sample processing system of the present embodiment performs the following processing steps by automatic operation.
(1) Destaining step of protein gel band stained with Coomassie blue dye.
(2) Gel band washing and dehydration process.
(3) A gel band drying step.
(4) A reductive alkylation step.
(5) Enzymatic digestion reaction process.
(6) A step of inspecting whether the steps (1), (4), and (5) are proceeding as expected.
(7) A step of recording the inspection result together with the sample identification number.
(8) A step that varies according to the inspection result.

  Details of each step will be described below. FIG. 8 is a flowchart showing a protein gel band destaining process.

  In the destaining process, the control unit 24 first moves the transport head 23 above the sample container storage 11 and uses the first container transport unit of the transport head 23 to prepare eight sample containers corresponding to one unit. The sample container 11 is taken out. The transport head 23 holding the sample container for one unit moves above the barcode reader 15 and reads the information of the two-dimensional barcode provided on the lower surface of each container by the barcode reader 15 (S21). The read information is stored in the memory of the control unit 24. Next, the transport head 23 transports the sample container to the dispensing station 14, opens the lid after lowering the container. In the dispensing station, the destaining solution (acetic acid methanol solution) is dispensed into each container constituting one unit (S22). The transport head closes the lid of the sample container into which the destaining liquid has been dispensed, and transports the sample container to the vibrating thermostat 16. Even in the following dispensing operation, the lid is opened immediately before dispensing unless otherwise specified, and after dispensing, the lid is closed immediately after that and other processing is performed.

  The temperature of the vibration thermostat 16 is adjusted to 37 ° C. ± 1 ° C., and the sample is incubated in the bath and washed by stirring (S23). When cleaning for a predetermined time is finished, one unit of the sample container is again transported to the dispensing station 14 by the transport head. In the dispensing station 14, a nozzle is inserted into each unit of the sample container to remove the destaining liquid by suction (S24). The sample container from which the destaining liquid has been removed is transported to the inspection station 20 by the transport head 23.

  A CCD camera is installed in the inspection station 20 as an imaging device. As shown in FIG. 9, the transport head 23 moves a unit row of containers in front of the CCD camera and samples samples in each container. Inspect (S25). In this inspection process, if the gel piece in the container is sufficiently destained, it is determined to be acceptable, and if the color tone of the gel piece is equal to or greater than a predetermined threshold, it is determined that the destain is insufficient and defective (S26). ).

  When the inspection is completed for all the containers, the transfer head 23 moves to the dispensing station, where one unit of the sample container is lowered. Next, the defective sample is transported to the place of the two-dimensional barcode reader 15 using the second container transport portion of the transport head 23, and the barcode information of the defective sample is read (S28). The read information is stored in the memory of the control unit 24. Thereafter, the container containing the defective sample is transported to the defective sample storage 13 by the transport head 23 for temporary retreat.

  Next, the transport head 23 uses the second container transport unit to take out the dummy container from the dummy container storage area 12 and remove it from the empty part of the container row remaining in the dispensing station (extracted as a defective sample). Insert the container into the place where there was a container. This operation is repeated as many times as the number of defective samples, and the number of containers placed in the dispensing station is set to 8 and unitized again. The dummy containers are added to form eight units, and the unitized containers are transported to the sample container storage 11 and wait for the next processing (S27).

  Next, a description will be given of the defect processing for a sample that fails the inspection.

  The control unit 24 first moves the transport head 23 above the defective sample storage 13 and picks up the container containing the unacceptable sample that has been evacuated to the defective sample storage 13 using the second container transport unit. And transport it to the dispensing station 14. This operation is repeated by the number of defective samples, and all containers containing defective samples are transported to the dispensing station 14. Next, a required number of dummy containers are transferred from the dummy container storage area 12 to the dispensing station 14 using the second container transfer section of the transfer head 23, and a unit consisting of eight containers is configured (S29). .

  Next, nozzles are inserted into all containers including the dummy containers to dispense the destaining solution (S22). When the destaining liquid has been dispensed, the unit is pulled up from the dispensing station 14 by the first container transport unit of the transport head 23 and transported to the vibration thermostat 16. The unit including the container containing the defective sample is incubated and destained in the vibration thermostat 16 (S23). When the destaining is completed, the sample container of the unit is transported to the dispensing station 14 by the transport head 23 again. In the dispensing station 14, a nozzle is inserted into each unit of the sample container to remove the destaining liquid by suction (S24). The sample container from which the destaining liquid has been removed is transported to the inspection station 20 by the transport head 23.

  In the inspection station, the transport head 23 moves the container row of one unit in front of the CCD camera, and the sample in the container is inspected (S25). At this time, since the position of the dummy container is known, the control unit does not inspect the dummy container using the information. When the necessary inspection is completed, the transfer head 23 moves to the dispensing station, where one unit of the sample container is lowered. If there is still a defective sample that has been rejected by the inspection, the defective sample is transported to the place of the two-dimensional barcode reader 15 using the second container transport portion of the transport head 23, and the barcode information of the defective sample is obtained. Read. The read information is stored in the memory of the control unit 24. Thereafter, the container containing the defective sample is transported to the defective sample storage 13 by the transport head 23.

  Next, the transport head 23 takes out the dummy container from the dummy container storage area 12 using the second container transport unit, and removes the dummy container from the container row remaining in the dispensing station (extracted as a defective sample). And insert it in the place where there was a container. In this way, the number of containers placed in the dispensing station is set to 8 and unitized again (S27). The number of dummy containers is added to make a unit, and the test-accepted sample containers are transported to the sample container storage 11 and wait for the next processing.

  The sample that has undergone the destaining process is then subjected to a washing and dehydrating process to remove the acetic acid methanol solution and facilitate drying. First, the control unit 24 moves the transport head 23 above the sample container storage 11, and uses the first container transport unit of the transport head 23 to remove eight sample containers corresponding to one unit from the sample container storage 11. Remove, transport to dispensing station 14 and lower container. In the dispensing station, ultrapure water is dispensed into each container constituting one unit. The transport head transports the sample container into which the cleaning liquid has been dispensed to the vibrating thermostat 16. The temperature of the vibrating thermostat 16 is adjusted to 37 ° C. ± 1 ° C., and the sample is incubated in the bath and stirred and washed. When cleaning for a predetermined time is finished, one unit of the sample container is again transported to the dispensing station 14 by the transport head. In the dispensing station 14, a nozzle is inserted into each of the sample containers of one unit to remove the cleaning liquid by suction. This cleaning process is performed a specified number of times.

  Next, the aqueous solution (acetonitrile) is dispensed into the sample container after washing. The transport head transports the sample container into which the dewatered solution has been dispensed to the vibrating thermostat 16. The temperature of the vibrating thermostat 16 is adjusted to 37 ° C. ± 1 ° C., and the sample is incubated in the bath for dehydration. When the processing for a predetermined time is completed, one unit of the sample container is again transported to the dispensing station 14 by the transport head. In the dispensing station 14, a nozzle is inserted into each of the sample containers of one unit to remove and remove the dewatered solution. After the dehydration process is performed a specified number of times, the sample containers are transferred to the sample container storage 11 for each unit, and wait for the next process.

  In the gel band drying step, one unit of the sample container placed on the sample container storage 11 is transported to the vacuum centrifuge 17 by the transport head 23 and vacuum-centrifuged for 30 minutes. The container of the unit after the drying process is returned to the sample container storage 11.

  FIG. 10 is a flowchart showing reductive alkylation treatment of a protein gel band.

  In the reductive alkylation step, the cysteine residue in the protein in the gel is reduced and then protected with an alkyl group. The reaction is carried out under a nitrogen atmosphere and under light-shielding conditions so that a reductive alkylation reaction using dithiothreitol as the reducing solution and iodoacetic acid as the alkylating agent is possible. First, a reducing solution is prepared (S41). Next, the control unit 24 drives the transport head 23 to transport one unit of sample container placed in the sample container storage 11 to the dispensing station 14. In the dispensing station 14, a reducing solution prepared in advance is dispensed into each conveyed container (S31). Next, the conveyance head 23 conveys the container of 1 unit in which the reducing solution was dispensed to the thermostat 18a. The container is left in the thermostatic chamber 18a (37 ° C., 30 minutes) to be reacted. Similarly, a reduction reaction is performed for all units (S32).

  Next, the transport head 23 transports an empty container from the container container 51 for inspection to the dispensing station 14 in preparation for a pH test. The pH indicator in the reagent storage 50 in the station is dispensed into this container (S42). When the reaction time is completed, one unit of the sample container is transferred from the thermostatic chamber 18a to the dispensing station 14, and a part of the reaction solution is sucked by the dispenser (S33). The aspirated reaction solution is discharged in the test container and mixed with the previously dispensed pH indicator by repeating the suction and discharge (S43).

  The inspection container is transported to the inspection station 20. The pH indicator color change is detected by the CCD camera of the inspection station 20. Before the sample analysis, the CCD camera records an image obtained by mixing a pH known buffer with a pH indicator for calibration, and the control unit makes a determination by comparing these with the actual measured value of the sample (S44).

  If there is a poor pH sample, the normal sample container of the unit is transported to the original location of the thermostatic chamber (S35). After reading the barcode on the defective sample container (S45), the remaining liquid is sucked (S46), and the reducing solution is again dispensed (100 μl) (S47). Then, it conveys to the original place of the thermostat 18a, leaves it in the thermostat 18a (37 degreeC, 60 minutes), and is made to react (S35).

  Subsequently, an alkylation solution is prepared (S48). When the reduction reaction time ends, the transport head 23 transports one unit of the container in the thermostat 18 a to the dispensing station 14. In the dispensing station 14, the gel-unabsorbed solution is completely aspirated from each container (36), and the alkylated solution is dispensed (100 μl) (S37). Thereafter, the transport head 23 transports the container to the light-shielding tank 19, leaves it (room temperature, 30 minutes), and causes it to react (S38).

  Next, the transport head 23 transports the container from the light shielding tank 19 to the inspection station 20 and performs a pH test in the same manner as in the reduction process. When there is a poor pH sample, after reading the barcode with the barcode reader (S52), the residual solution is aspirated (S53), and the reducing solution is dispensed (100 μl) (S54). Then, it is transported to the ice tub 18b and waited (S55). These samples are subjected to reductive alkylation reaction again from S32 after the work of normal samples is completed.

  The sample that has undergone the reductive alkylation reaction step is then subjected to a washing and dehydration step for removing excess reagents and buffers. This process is the same as the above-described washing and dehydrating processes except that an ammonium bicarbonate solution is used instead of ultrapure water.

  The sample that has been subjected to the washing and dehydration process is again subjected to the drying process as described above.

  Next, the enzyme digestion reaction step will be briefly described. First, an enzyme solution is prepared. The proteolytic enzyme is prepared in advance, and is put in a container as a lyophilized product in an amount necessary for one analysis in the reagent storage 50 in the dispensing station. In this container, ultrapure water is dispensed to a prescribed concentration by a dispenser, and suction and discharge are repeated and dissolved. Next, one unit of the sample container placed in the sample container storage 11 is transported to the dispensing station 14. After dispensing a predetermined amount of the previously prepared enzyme solution into each sample container, it is conveyed to the ice bath 18b. The container is placed in an ice bath and the gel in the container is swollen for a predetermined time. A container containing the gel swollen for a predetermined time is transported to an inspection station, and the degree of swelling is inspected. The dried gel before swelling is white, whereas the gel swollen by absorbing the enzyme solution becomes colorless and transparent. Therefore, the degree of swelling is determined using this decrease in white as an index.

  One unit of the sample container for which the swelling test is completed is transported to a dispensing station. If there is a poorly swelled sample, the defective sample is transported to the place of the two-dimensional barcode reader 15 using the second container transport unit of the transport head 23, and the barcode information of the defective sample is read. The read information is stored in the memory of the control unit 24. Thereafter, the container containing the defective sample is transported to the ice tub 18b by the transport head 23 for temporary retreat.

  Next, the transport head 23 uses the second container transport unit to take out the dummy container from the dummy container storage 12 and remove it as a vacant part of the container row remaining in the dispensing station (as a defective sample). Insert the container into the place where there was a container. This operation is repeated as many times as the number of defective samples, and the number of containers placed in the dispensing station is set to 8 and unitized again. Dispense the digestion buffer into this unit, transport it to the ice bath again, and leave it for a predetermined time.

  After the predetermined time has passed, the sample container is transported to a dispensing station, and a prescribed amount of digestion buffer is added again. The transport head 23 transports the sample container to which the digestion buffer is added to the thermostatic chamber 18a. The temperature of the thermostatic chamber is adjusted to 37 ° C. ± 1 ° C., and the enzyme digestion reaction is performed by leaving the sample in the bath. Next, the transport head 23 transports an empty container from the container container 51 for inspection to the dispensing station 14 in preparation for a pH test. The pH indicator in the reagent storage 50 in the station is dispensed into this container. When the reaction time is completed, one unit of the sample container is transferred from the thermostatic bath 18a to the dispensing station 14, and a part of the reaction solution is sucked by the dispenser. The aspirated reaction liquid is discharged in a test container and mixed with a previously dispensed pH indicator by suction and discharge. The inspection container is transported to the inspection station 20. The pH indicator color change is detected by a CCD camera. Before the sample analysis, the CCD camera records an image obtained by mixing a buffer solution having a known pH with a pH indicator for calibration, and the control unit 24 makes a determination by comparing these with the actual measured value of the sample.

  In the above description, in order to explain a general operation, each process is started from the sample container storage place and then returned to the sample storage place. However, when a specific protocol is actually executed, the respective steps can be directly connected without using a sample storage place. For example, a reducing solution can be prepared during the drying process, and when the drying process is completed, the reducing solution can be dispensed by transferring one unit directly from the vacuum centrifuge to the dispensing station. In this way, more efficient preparation is possible by directly combining the steps without using the sample storage place.

  In the above embodiment, each sample container is assigned a barcode, and the sample and information are managed by the barcode. FIG. 11 is an explanatory diagram showing an example of using barcode data.

  The management of the sample container is based on the two-dimensional barcode and the physical position at the sample storage. In this embodiment, the barcode information is attached to the bottom surface of the container in which the sample is placed. For the position data, the number of the 8 units is the row number (for example, when preparing 96 samples, the row number is 1 to 12), and the position of the 8 units is the column number. 2D data. For example, the fifth tube from the origin of the third set is registered as 3-5 (see FIG. 11A).

  FIG. 11B shows an example of how bar code information and position information are managed. At the beginning of automatic operation, the transport head transports the sample containers on a bar code reader for each unit (eight) to read the bar code of each container. The control unit 24 generates a new data line every time the barcode is read, and records position information in the initial position field and the current position field as shown in FIG. During the automatic operation, the sample container is taken out for each unit, processed, and returned to the original position of the sample storage area 11, so that the relationship between the barcode information and the position information is maintained.

  When it is determined as a defective sample in the inspection process, the barcode is read, and a failure (false) is recorded in the corresponding inspection result column. The container containing the rejected sample is transported to a predetermined place such as a defective sample storage place, and the dummy container is transported to the original position instead. The current position column of the control unit is rewritten to the position where the defective sample is conveyed. When the initial position field and the current position field are different, the control unit recognizes that there is a dummy container in the corresponding initial position field and performs the subsequent steps.

1 is a schematic diagram showing the overall configuration of a sample processing system according to the present invention. 4 is an explanatory diagram illustrating an example of a transport head. FIG. Explanatory drawing which shows an example of the conveyance head provided with the dispensing apparatus. Explanatory drawing which shows the mode of the container conveyance by a rod provided with a container holding part at the front-end | tip. The flowchart which shows the outline of the process in each process process. The conceptual diagram which showed the flowchart of FIG. 4 visually. Explanatory drawing which showed in detail the flow of the container containing the sample which passed the inspection, and the container containing the rejected sample. The flowchart which shows the destaining process of a protein gel band. The figure explaining an example of the inspection method. The flowchart which shows the reductive alkylation process of a protein gel band. Explanatory drawing which shows management of the sample container information by a barcode.

Explanation of symbols

11: Sample container storage area, 12: Dummy container storage area, 13: Defective sample storage area, 14: Dispensing station, 15: Bar code reader, 16: Vibrating thermostatic chamber, 17: Vacuum centrifuge, 18a: Thermostatic bath, 18b: Ice Bathtub, 19: Shading tank, 20: Inspection station, 21a, 21b: Rail, 22: Ball screw, 23: Transfer head, 24: Control unit, 31: Rod, 32: Container gripping unit, 33: First container transfer unit , 34: second container transport unit, 41: sample container, 50: reagent bottle storage area, 51: inspection container storage area, 52: rod outer cylinder, 53: container lid, 54: holding pin, 55: dispenser head, 56: Outer cylinder for chip detachment, 57: Pipette tip, 58: First dispensing section, 59: Second dispensing section, 60: Pipette tip storage place, 61: Pipette tip disposal place

Claims (12)

In a multiple sample automatic processing system that processes multiple samples through multiple processing steps,
A sample container storage place where a plurality of sample containers are placed;
A dummy container yard where dummy containers are placed;
A first processing unit and a second processing unit for performing different processing on the sample in the container;
An inspection unit for inspecting a sample in a container;
A container transport section for transporting the container between the container storage place, the processing section and the inspection section;
A control unit,
The control unit controls each of the units so that n units (n is a positive integer greater than or equal to 2) are processed as one unit in the first processing unit. When there is a sample that has been accepted, a dummy container is added to the container containing the sample that has been rejected, and the n-paired container is subjected to defect processing, and the container containing the sample that has passed the inspection. A multiple sample automatic processing system, wherein a dummy container is added to the container to process a set of n containers in the second processing section.   2. The automatic multiple sample processing system according to claim 1, wherein in the defect processing, the set of n containers is returned to the first processing unit for reprocessing. 3.   The multiple sample automatic processing system according to claim 1 or 2, wherein the container transport unit includes a first container holding unit that holds n containers simultaneously and a second container holding unit that holds one container. A multiple sample automatic processing system comprising:   The multiple sample automatic processing system according to any one of claims 1 to 3, wherein the first processing unit and the second processing unit inject or suck a processing liquid into the n sets of containers. An automatic multiple sample processing system comprising dispensing means.   5. The multiple sample automatic processing system according to claim 4, wherein the dispensing means is shared by the first processing unit and the second processing unit.   6. The multiple sample automatic processing system according to claim 5, wherein the dispensing means is provided in the container transport unit.   The multiple sample automatic processing system according to any one of claims 1 to 6, wherein the inspection unit includes an imaging device and images and inspects a sample in each container. system. In a multiple sample automatic processing method for processing a plurality of samples through a plurality of processing steps,
In the first processing unit, a process of processing samples in the container as a set of n containers (n is a positive integer of 2 or more);
Performing a first inspection on a sample that has been processed by the first processing unit;
A step of forming a first container unit composed of n containers by adding a dummy container to a container containing the rejected sample when there is a sample rejected by the first inspection;
Performing a defect treatment on the first container unit;
Performing a second inspection on the sample after the defect processing;
Adding a dummy container to a container containing a sample that has passed the second inspection to form a second container unit consisting of n containers;
Adding a dummy container to a container containing a sample that has passed the first inspection to form a third container unit comprising n containers;
And a step of processing the sample in the second container unit and the sample in the third container unit in the second processing unit.   9. The multiple sample automatic processing method according to claim 8, wherein the first container unit includes a mixture of containers that have failed in the first inspection for different sets of containers. A method for automatically processing multiple samples.   10. The multiple sample automatic processing method according to claim 8, wherein in the defect processing, the first container unit is returned to the first processing unit for processing.   The multiple sample automatic processing method according to any one of claims 8 to 10, wherein in the first processing section, a processing liquid is injected into the n containers to cause a reaction, and the processing liquid is sucked after the reaction. A method for automatically processing a plurality of samples.   The automatic multiple sample processing method according to any one of claims 8 to 11, wherein the inspection is performed by imaging a sample in the container.

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